For the production of soy sauce, miso, and other natural seasonings containing protein hydrolysate products, koji mould (filamentous fungus belonging to the genus Aspergillus) has been utilized. For example, soy sauce is produced through two process steps of koji-making and fermentation. In the koji-making step, the starting material is principally degraded by enzymes produced by koji mould. In such a process, it is important to increase the amount of glutamic acid among various tasteful materials in order to obtain stronger umami of soy sauce.
Glutamic acid is produced through two kinds of pathways. The first is the liberation of glutamic acid from protein caused by protease and peptidase. The second is generation of glutamic acid through hydrolysis of glutamine catalyzed by glutaminase (glutamine amidohydrolase).
In the production of soy sauce, liberation ratio of glutamic acid relative to its content in the starting material is not so high, and this is considered to be due to insufficient glutaminase activity of koji mould. Therefore, breeding of strains exhibiting high activities of protease and glutaminase through cell fusion of high protease activity strain and high glutaminase activity strain in solid koji has also been attempted (Ushijima, S. et al., Agric. Biol. Chem., 51 (4), 1051 (1987), Japanese Patent Publication (KOKOKU) No. Hei 3-73271/1992).
As for glutaminase, those derived from various bacteria and animals have been well investigated (Wakayama, M. et al., J. Ferment. Bioeng., 82, No.6, 592–597 (1996), Chung-Bok, Mi, et al., Biochem. J., 324, 193–200 (1997), Duran, S. et al., Biochem. Genet., 34, 453–465 (1996)). On the other hand, investigation about glutaminase of koji mould had been retarded, but extracellular glutaminase and intracellular glutaminase have been purified from one strain of Aspergillus oryzae, and they have been characterized (Yano, T. et al., J. Ferment. Technol., Vol. 66, No. 2, 137–143 (1988)). These glutaminases have a molecular weight of about 113,000, and substantially similar properties.
Further, there have been determined an amino acid sequence of N-terminal region of glutaminase derived from Aspergillus oryzae HG strain (Fukuoka Industrial Technology Center, Institute of Biology and Food, Research Summary of 1996 (199)), and amino acid sequence within N-terminal region of glutaminase derived from Aspergillus oryzae (Food Research Institute, Aichi Prefectural Government, Japan, Annual Report of 1995 (Research Report) pp. 3–4, (1996)) for purified glutaminases.
Meanwhile, because koji mould is excellent in the ability for secreting extracellular proteins, it has been attracted attention as a host for the production of recombinant proteins, and practically used for some enzymes.